The following chemicals and reagents were purchased from the indicated sources: Protease type XIV, 5-[N-2,3-dihydroxypropylacetamido-2,4,6-triiodo-N,N′-bis(2,3-dihydroxypropyl)isophthalamide] (Nycodenz), BQ-788 (N-cis-2,6-dimethylpiperidinecarbonyl-L-γMeLeu-D-Trp(COOMe)-D-Nle-ONa) and 13-cis-retinoic acid (Sigma Chemical Co., St Louis, MO, U.S.A.); collagenase type IV (Worthington Biochemical Corporation, Freehold, NJ, U.S.A.); cell culture media and sera (GIBCO-BRL, Grand Island, NY, U.S.A.); ET-1, sarafotoxin S6c and BQ-123 (cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu-)) (American Peptide, Sunnyvale, CA, U.S.A.); [125I]TYR13-endothelin-1 (2200 Ci mmol−1) (Perkin-Elmer-New England Nuclear, Boston, MA, U.S.A.); [Methyl-3H]thymidine (88.7 Ci mmol−1) (Perkin-Elmer Life Sci., Boston, MA, U.S.A.); and L-[2,3,4,5-3H]proline (100 Ci mmol−1) (Amersham-Pharmacia, Piscataway, NJ, U.S.A.). Retinoic acid was dissolved in ethanol at 5 mM concentration and stored protected from light in aliquots under nitrogen. Fresh solution was made every week, and the cell culture work was performed protected from the direct light. The solution of retinoic acid or ethanol was added at 1 μl ml−1 to the cultured cells. When ET-1, sarafotoxin S6c, or the ET receptor antagonists were added to the incubations, equivalent volume of the solvent (phosphate-buffered saline (PBS)) was added to the control cells.
Preparation of stellate cells
The experimental protocols were approved by the University of Pittsburgh IACUC in accordance with the guidelines of the National Institutes of Health. HSC were prepared essentially as described previously (Uemura & Gandhi, 2001). Briefly, the livers of male Sprague–Dawley rats (450–500 g) were digested with protease and collagenase. Hepatocytes and cell debris were removed by low-speed centrifugation (50 × g for 1 min; 2 ×), and the supernatant was centrifuged at 1400 × g for 7 min. The pellet (nonparenchymal cells) of this centrifugation was suspended in Hank's balanced salt solution (HBSS) and subjected to the above steps to remove residual hepatocytes and cell debris. HSC were purified from the other nonparenchymal cells by centrifugation (1400 × g for 20 min) on a 13.14% (w/v) Nycodenz gradient. The cells were suspended in DMEM containing 10% fetal bovine serum (FBS)/10% horse serum and antibiotics, and plated in 24-well or six-well culture plates (0.1 × 106 cells/cm2). The viability of the cells as determined by trypan blue exclusion was greater than 97%. After overnight incubation, the cells were washed and placed in DMEM containing 2.5% FBS/2.5% horse serum with or without 2.5 μM 13-cis-retinoic acid. The medium was renewed every day and experiments were performed between days 10 and 12. The purity of the cells, as determined by vitamin A autofluorescence and immunohistochemical analysis for desmin (Gandhi et al., 2000; Uemura & Gandhi, 2001), was greater than 95%.
[125I]ET-1 binding assay
Cells were washed with HBSS containing 10 mM HEPES, pH 7.4, and 0.1% bovine serum albumin (HBSS/BSA), and placed in this medium containing 6.25–800 pM [125I]ET-1 in the absence or presence of 1 μM unlabeled ET-1 (saturation binding). In the competition binding assay, cells were incubated with 20 pM [125I]ET-1 in the absence or presence of 1 μM unlabeled ET-1, 10 pM–10 μM ETA antagonist BQ-123 or 10 pM–10 μM ETB agonist sarafotoxin S6c. After incubation at 22°C for 2 h, the cells were washed with HBSS/BSA and digested with 0.75 N NaOH for determination of radioactivity. Specific binding of [I25I]ET-1 was calculated as the difference between cell-associated radioactivity in the presence and absence of 1 μM unlabeled ET-1.
Determination of mRNA expression
Semiquantitative reverse transcriptase–polymerase chain reaction (RT–PCR) was performed to assess the relative mRNA expressions of preproET-1, ET-1 receptors, collagen α type-I, collagen α type-III and TGF-β1 as described previously (Gabriel et al., 1999; Gandhi et al., 2000). The PCR primers specific for preproET-1 cDNA: 5′CCAACTCTGGGCTCTCCATGCT-GG3′ (F) and 5′GAATGGCACTGTGTCTCTG-CTCTC3′ (R) [241 bp]; ETA cDNA: 5′CCCTTCGAATACAAGGGCGA3′ (F) and 5′GAAGAGGGAACCAGCAC3′ (R) [291 bp]; ETB cDNA: 5′GGCTGTTCAGTTTCTAC-TTCTGC3′ (F) and 5′AGAA-TCCTGCTGAGGTGAAGG3′ (R) [210 bp]; TGF-β1 cDNA: 5′AGCTCAGACATTCGGGA-AGCAGTG3′ (F) and 5′GC-AAGGACCTTGCTGTACTG-TGTG3′ (R) [615 bp]; collagen α type-I cDNA: 5′GGTTCTCGGACTATTGAAGGAGC3′ (F) and 5′AGACAA-GAACGAGGTAGTCTTTC3′ (R) [245 bp]; collagen α type-III cDNA: 5′CGAGGTAACAGAGGTGAA-AGA3′ (F) and 5′AACCCAGTATTCTCCGCTCTT3′ (R) [349 bp]; and β-actin cDNA: 5TTCTACAATGAGCTGC-GTGTG3′ (F) and 5′TTCATGGATGCCACAGGATTC3′ (R) [561 bp] were used. The PCR products were resolved in a 1.2% agarose gel (β-actin and TGF-β1) or 1.7% agarose gel (ET-1, ETA, ETB, collagen I and collagen III), and stained with SYBR Green I (FMC Biproduct, Rockland, ME, U.S.A.). The gels were scanned under blue fluorescence light using a phosphorimager and the band intensity was quantified using ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA, U.S.A.). The expression of various mRNAs was normalized with respect to that of β-actin mRNA by calculating the ratio of band intensities.
The morphology of the cells was determined by phase-contrast microscopy with an Olympus CK2 microscope. For detailed analysis, single confocal slices were taken with an Olympus Fluoview Confocal microscope (Melview, NY, U.S.A.), and differential interference contrast images were overlayed with the images of the same cells stained with nuclear sytox green (Molecular Probes, Eugene, OR, U.S.A.). For determination of cell size, all measurements of maximum fiberlength were done manually using Metamorph version 6.03 program (Universal Imaging Corp., Downington, PA, U.S.A.). The Metamorph region tools were used to show maximum dimensional length of the cells. This length was then logged and all dimensions were calibrated on the microscope using a linear standard line. This calibration was applied to each image to ensure reproducibility between experiments. Images of 20–30 randomly chosen cells from replicate wells (three experiments for each condition with different batches of cells) were analyzed. The maximum length of individual cell was considered for final determination of average cell length.
Western blot analysis for α-sma
Cells in six-well plates were washed, placed in 50 μl lysis buffer (0.1 M.NaCl, 10 mM Tris-HCl, 1 mM EDTA) containing 0.5 mM PMSF and 25 μl ml−1 protease inhibitor cocktail (P-8340; Sigma Chemical Co.) for 10 min on ice, and scraped. The lysate was stored in aliquots at –80°C until use. Proteins (15 μg) from HSC lysates were mixed with 2 × loading buffer (5% SDS, 0.125 M Tris-HCl, pH 7.4, 0.03 M EDTA, 20% glycerol, 3% DL-dithiothreitol and 0.01% bromophenol blue) and heated at 100°C for 3 min, then cooled on ice. Proteins were separated by electrophoresis on an 8% SDS–PAGE minigel and transferred onto an Immobilon-P membrane (Millipore Corp., Bedford, MA, U.S.A.) in a Mini-Trans Blot transfer system (Bio-Rad Laboratories). The membrane was rinsed briefly in PBS, and equal loading was confirmed by staining with Ponceau S. The stain was removed from the membrane by a brief wash with water, after which the nonspecific binding was blocked by incubation in 5% nonfat milk in PBS for 2 h. The membrane was then washed in PBS for 15 min (4 ×), incubated overnight at 4°C with a monoclonal anti-α-SMA antibody (Sigma Chemical Co., 1 : 1000 dilution) in 0.1% nonfat milk/PBS, washed with PBS for 15 min (2 ×) and for 5 min (2 ×), incubated for 2 h with rabbit anti-mouse IgG (peroxidase conjugate, Sigma Chemical Co.) (1 : 4000 in 0.1% nonfat milk/PBS). After washing the membrane with PBS for 15 min (4 ×), detection was achieved using an ECL chemiluminescence kit (Amersham-Pharmacia). The intensity of the bands was determined by densitometry using ImageQuaNT software (Molecular Dynamics). To normalize the data, the same amount of protein was separated on another gel and the level of expression of a nonvariant protein Erk 2 was determined using a polyclonal antibody (clone C-14) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.).
The synthesis of collagen was determined by the procedure described previously (Shina et al., 1989; Gandhi et al., 2000). Cells were washed and placed in DMEM/0.1% BSA containing 5 μCi ml−1 [3H]proline and various agents at concentrations indicated in the figure legends. After incubation for 24 h at 37°C, the medium was aspirated, mixed with chick embryo extract (GibcoBRL) (50 μl ml−1) and proteins were precipitated with 10% TCA. After centrifugation, the pellet was washed twice with 10% TCA and dissolved in 0.6 ml 0.2 N NaOH. An aliquot (0.2 ml) of this solution was neutralized with HCl, mixed with 0.1 ml 50 mM Tris-Cl, pH 7.4, containing 5 mM CaCl2 and 10 mMN-ethylmaleimide, and the reaction was stimulated with 20 U ml−1 collagenase (type VII from Clostridium histolyticum, Sigma Chemical Co.). The total volume of the reaction mixture was 0.5 ml. After incubation for 3 h at 37°C, 50 μl of 10 mg ml−1 BSA and 50 μl of 100% TCA were added to each tube. After centrifugation, an aliquot of the supernatant was aspirated for determination of radioactivity. Negative controls (i.e. without enzyme) were used in each assay for determination of nonspecific release of [3H]proline.